This proposal considers the hypothesis that the interactions of a phospholipase C (PLC)-mediated pathway, and a Ca2+-dependent phospholipase A2 (PLA2) pathway play a pivotal role in stimulus-secretion coupling in exocrine glands. These studies will be performed in isolated rat pancreatic acini, as well as intact or permeabilized acinar cells, prelabeled with radiolabeled precursors (e.g. 3H-inositol, 14C-arachidonic acid, 32P) to label phospholipids, inositol polyphosphates, arachidonic acid metabolites, and phosphoproteins. We will investigate the relative abilities of phorbol esters and diacylglycerol analogues, which directly activate protein kinase C, to interact synergistically with activators of PLA2 (ionomycin, caerulein, carbachol) with regard to enhancing cellular [Ca2+], arachidonic acid metabolism, protein phosphorylation, and amylase secretion. We will examine the comparative abilities of pancreatic secretagogues (carbachol, caerulein, ionomycin) to stimulate the formation of inositol phosphates, mobilize cellular Ca2+, and activate PLA2 in intact and permeabilized cells. 32P-IP3 binding to a specific cellular receptor site will be characterized in permeabilized cells and subcellular fractions. The concentration dependence of 1,4,5-IP3-induced 45Ca release will also be probed to delineate physiological significance of the binding data. To examine the relative contributions of PLA2 and PLC pathways in the release of arachidonic acid from inositol phospholipids, the identification and activation of diglyceride lipase, phosphatidic acid (PA)-specific PLA2 activities, as well as lysophospholipid (lysoPA, lysoPI) accumulation will be investigated in stimulated acini. The extent and distribution of lysophospholipid acylation activities in purified subcellular fractions of pancreatic acini will also be examined using arachidonic acid metabolites (HETEs, leukotrienes) as donors. The effects of lipoxygenase products on basal and stimulated PLC-mediated reactions and amylase secretion will also be examined. Finally, characterization of the profile of arachidonic acid metabolites (particularly lipoxygenase products) will be carried out by HPLC, RIA, and gas chromatography/mass spectrometry to attribute physiological relevance to the biochemical studies. These studies should render support for the association between inositol phospholipid breakdown mediated by PLC and PLA2, CA2+ metabolism, protein phosphorylation, and amylase secretion.